TMidbrain dopamine (DA) neurons play a role in reward, motivation, learning, memory, and movement. Midbrain DA neurons, containing tyrosine hydroxylase (TH), are organized into three cell groups: A8 (retrorubral area, RRF), A9 (substantia nigra, SN), and A10 ventral tegmental area (VTA), rostral linear nucleus of the raphe (RLi), caudal linear nucleus of the raphe (CLi), and interfascicular nucleus (IF). In addition to DA neurons, gamma-amino butyric acid (GABA) neurons are present in the midbrain DA system. Moreover, we had previously reported the presence of glutamatergic neurons expressing the vesicular glutamate transporter 2 (vGluT2) in all subdivisions of the midbrain DA system. We had also shown that a subpopulation of vGluT2 neurons confined to the medial portion of the midbrain DA system contains TH (Yamaguchi et al, SfN 2008). Here, by applying in situ hybridization in combination with immunohistochemistry, we analyzed the rostro-caudal and medio-lateral distribution within the midbrain DA system of the two subpopulations of vGluT2 neurons: vGluT2 neurons lacking TH (vGluT2) and vGluT2 neurons co-expressing TH (vGluT2/TH). We found that vGluT2 neurons of the SN pars compacta (SNC) were preferentially located in the lateral portion of this structure. Within the SNC, the highest concentration of vGluT2 cells was detected from bregma -5.52 mm to -6.12 mm with a ratio of 4.4 TH neurons for each vGluT2 neuron. The highest concentration of vGluT2 cells within the SN pars lateralis (SNL) and the SN pars reticulata (SNR) was found in the caudal levels of these structures, with a ratio of 3.2 TH cells for each vGluT2 in the SNR, and 1.1 TH cell for each vGluT2 in the SNL. Throughout the RRF, vGluT2 neurons were intermingled with DA neurons, and a ratio of 1.7 TH neurons for each vGluT2 neuron was maintained from the rostro-caudal aspects of the RRF. In agreement with our previous studies (Yamaguchi et al, SfN 2008, see also Li et al., SfN 2010), we did not detect vGluT2 mRNA in TH cells located within the SNC, SNL or RRF. In a follow up study, we determined the distribution and proportion of the two subpopulations of vGluT2 neurons in the medial region of the midbrain. The ratio of these two subtypes of neurons was 0.28 vGluT2/TH neuron for each vGluT2 neuron in the RLi, 0.19 vGluT2/TH neurons for each vGluT2 neuron in the CLi, and 0.31 vGluT2/TH neuron for each vGluT2 neuron in the IF. In summary, 1) glutamatergic neurons are not homogeneously distributed within all subdivisions of the midbrain DA system, 2) glutamatergic neurons expressing TH are enriched in the medial portion of the midbrain, 3) the lateral part of the midbrain contains exclusively glutamatergic neurons lacking TH.